An enigmatic peptide ligation reaction: protease-catalyzed oligomerization of a native protein segment in neat aqueous solution

We report an enigmatic peptide ligation reaction catalyzed by Glu-specific Staphylococcus aureus V8 protease that occurs in neat aqueous solution around neutral pH utilizing a totally unprotected peptide substrate containing free alpha-carboxyl and alpha-amino groups. V8 protease catalyzed a chain of ligation steps between pH 6 and 8 at 4 degrees C, producing a gamut of covalent oligomers (dimer through octamer or higher) of a native protein segment TAAAKFE (S39) derived from ribonuclease A (RNAse A). Size-exclusion chromatography suggested the absence of strong interaction between the reacting peptides. The circular dichroism spectra of monomer through pentamer showed length-dependent enhancement of secondary structure in the oligomers, suggesting that protease-catalyzed ligation of a monomer to an oligomer resulted in a product that was more structured than its precursor. The relative conformational stability of the oligomers was reflected in their ability to resist proteolysis, indicating that the oligomerization reaction was facilitated as a consequence of the "conformational trapping" of the product. The ligation reaction proceeded in two phases-slow formation and accumulation of the dimer followed by a fast phase of oligomerization, implying that the conformational trap encountered in the oligomerization reaction was a two-step process. The Gly substitution at any position of the TAAAKFE sequence was deleterious, suggesting that the first step of the conformational trap, namely the dimerization reaction, that proceeded very slowly even with the parent peptide, was quite sensitive to amino acid sequence. In contrast, the oligomerization reaction of an Ala analog, AAAAKFE, occurred in much the same way as S39, albeit with faster rate, suggesting that Ala substitution stabilized the overall conformational trapping process. The results suggest the viability of the product-directed "conformational trap" as a mechanism to achieve peptide ligation of totally unprotected peptide fragments in neat aqueous solution. Further, the study projects the presence of considerable innate synthetic potential in V8 protease, baring rich possibilities of protein engineering of this enzyme to generate a "V8 peptide ligase."     

Application of quantitative structure‐activity relationship analysis on an antibody and alternariol‐like compounds interaction study

The antigen‐antibody interaction determines the sensitivity and specificity of competitive immunoassay for hapten detection. In this paper, the specificity of a monoclonal antibody against alternariol‐like compounds was evaluated through indirect competitive ELISA. The results showed that the antibody had cross‐reactivity with 33 compounds with the binding affinity (expressed by IC₅₀) ranging from 9.4 ng/mL to 12.0 μg/mL. All the 33 compounds contained a common moiety and similar substituents. To understand how this common moiety and substituents affected the recognition ability of the antibody, a three‐dimensional quantitative structure‐activity relationship (3D‐QSAR) between the antibody and the 33 alternariol‐like compounds was constructed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods. The q² values of the CoMFA and CoMSIA models were 0.785 and 0.782, respectively, and the r² values were 0.911 and 0.988, respectively, indicating that the models had good predictive ability. The results of 3D‐QSAR showed that the most important factor affecting antibody recognition was the hydrogen bond mainly formed by the hydroxyl group of alternariol, followed by the hydrophobic force mainly formed by the methyl group. This study provides a reference for the design of new hapten and the mechanisms for antibody recognition.     

Overview of the diversity of erythrocyte blood group antigens

During the differentiation and maturation of erythrocytes, the surface molecules of erythrocytes are gradually expressed and stabilized. These molecules are to be antigenic in addition to their functions of maintaining cell membrane structural stability, material transport and exchange of cells and signal transmission between cells. The antigenic molecules on the erythrocyte surface are called erythrocyte blood group antigens. The blood group antigens and their corresponding blood group antibodies in vivo are important indicators for clinical blood transfusion and organ transplantation, and also form the basis for research on blood group related diseases. Three hundred and sixty-eight erythrocyte blood group antigens have been confirmed so far, which are classified into 39 blood group systems, 5 blood group collections and 2 blood group series. Based on the diversity of blood group antigens and their composition of glycolipids, glycoproteins and other molecules, this study mainly reviews the classification, molecular structure, antibody response and gene regulation of blood group antigens, and explains the main reasons for the diversity of blood group antigens.     

生物合成谷胱甘肽种间耦合ATP再生系统的构建

利用重组大肠杆菌Ⅱ 1中的谷胱甘肽合成酶系和面包酵母WSH J7中的ATP生物合成酶系 ,构建了一个以葡萄糖为能源的种间耦合ATP再生系统。经过通透性处理的酵母细胞几乎不能消耗葡萄糖。在反应体系中添加 1mmol/LAMP和 0 0 5mmol/LNADH ,即可启动酵母的酵解途径。提高耦合系统中的葡萄糖浓度 ,可促进GSH的合成。当葡萄糖浓度为 40 0mmol/L时 ,系统内GSH浓度达到 1 0 4mmol/L(3 2 g/L)。Mg2 +缺乏时 ,耦合系统和外加ATP的非耦合系统均不能合成谷胱甘肽。耦合系统中Mg2 +与ATP形成螯合物 ,可能是导致耦合系统中GSH产量较低的原因。在耦合系统中补加Mg2 +,反应 6h时GSH浓度达到 1 4 3mmol/L(4 4g/L)。     

Identification of genomic regions that affect grain-mould incidence and other traits of agronomic importance in sorghum

Grain-mould is a major problem in grain sorghum utilization as mouldy grain has a reduced quality due to the deterioration of the endosperm and reduced embryo viability. Here, our objective was to use genome mapping to improve knowledge of genetic variation and co-variation for grain-mould incidence and other inter-related agronomic traits. Grain-mould incidence, kernel-milling hardness, grain density, plant height, panicle peduncle length, foliar-disease incidence, and plant color were measured on 125 F₅ genotypes derived from a cross of Sureño and RTx430. Quantitative trait loci were detected by means of 130 mapped markers (44 microsatellites, 85 AFLPs, one morphological-trait locus) distributed among ten linkage groups covering 970 cM. One to five QTLs affected each trait, with the exception of grain density for which no QTLs were detected. Grain-mould incidence was affected by five QTLs each accounting for between 10 and 23% of the phenotypic variance. The effects and relative positions of QTLs for grain-mould incidence were in accordance with the QTL distribution of several inter-related agronomic traits (e.g., plant height, peduncle length) and with the correlation between these phenotypic traits and grain-mould incidence. The detection of QTLs for grain-mould incidence was dependent on the environment, which is consistent with heritibility estimates that show strong environmental and genotype × environment effects. Several genomic regions affected multiple traits including one region that affected grain-mould incidence, plant height, panicle peduncle length, and grain-milling hardness, and a second region that influenced grain-mould (in four environments) and plant height. One genomic region, which harbors loci for plant color, influenced the severity of foliar disease symptoms and the incidence of grain-mould in one environment. Collectively QTLs detected in the present population explained between 10% and 55% of the phenotypic variance observed for a given trait.     

Assembly Pathway and Characterization of the RAG1/2-DNA Paired and Signal-end Complexes

Mammalian immune receptor diversity is established via a unique restricted set of site-specific DNA rearrangements in lymphoid cells, known as V(D)J recombination. The lymphoid-specific RAG1-RAG2 protein complex (RAG1/2) initiates this process by binding to two types of recombination signal sequences (RSS), 12RSS and 23RSS, and cleaving at the boundaries of RSS and V, D, or J gene segments, which are to be assembled into immunoglobulins and T-cell receptors. Here we dissect the ordered assembly of the RAG1/2 heterotetramer with 12RSS and 23RSS DNAs. We find that RAG1/2 binds only a single 12RSS or 23RSS and reserves the second DNA-binding site specifically for the complementary RSS, to form a paired complex that reflects the known 12/23 rule of V(D)J recombination. The assembled RAG1/2 paired complex is active in the presence of Mg²⁺, the physiologically relevant metal ion, in nicking and double-strand cleavage of both RSS DNAs to produce a signal-end complex. We report here the purification and initial crystallization of the RAG1/2 signal-end complex for atomic-resolution structure elucidation. Strict pairing of the 12RSS and 23RSS at the binding step, together with information from the crystal structure of RAG1/2, leads to a molecular explanation of the 12/23 rule.     

Vascular-homing peptides for targeted drug delivery and molecular imaging: Meeting the clinical challenges

The vasculature of each organ expresses distinct molecular signatures critically influenced by the pathological status. The heterogeneous profile of the vascular beds has been successfully unveiled by the in vivo phage display, a high-throughput tool for mapping normal, diseased, and tumor vasculature. Specific challenges of this growing field are targeted therapies against cancer and cardiovascular diseases, as well as novel bioimaging diagnostic tools. Tumor vasculature-homing peptides have been extensively evaluated in several preclinical and clinical studies both as targeted-therapy and diagnosis. To date, results from several Phase I and II trials have been reported and many other trials are currently ongoing or recruiting patients. In this review, advances in the identification of novel peptide ligands and their corresponding receptors on tumor endothelium through the in vivo phage display technology are discussed. Emphasis is given to recent findings in the clinical setting of vascular-homing peptides selected by in vivo phage display for the treatment of advanced malignancies and their altered vascular beds.     

Incorporation of the Tat cell‐penetrating peptide into nanofibers improves the respective antitumor immune response

A major challenge for the development of anticancer vaccines is the induction of a safe and effective immune response, particularly mediated by CD8+ T lymphocytes, in an adjuvant‐free manner. In this respect, we present a simple strategy to improve the specific CD8+ T cell responses using KFE8 nanofibers bearing a Class I (Kb)‐restricted peptide epitope (called E. nanofibers) without the use of adjuvant. We demonstrate that incorporation of Tat, a cell‐penetrating peptide (CPP) of the HIV transactivator protein, into E. nanofibers remarkably enhanced tumor‐specific CD8+ T cell responses. E. nanofibers containing 12.5% Tat peptide (E.Tat_12.5 nanofiber) increased antigen cross‐presentation by bone marrow‐derived dendritic cells as compared with E. nanofibers, or E. nanofibers containing 25 or 50% the Tat peptide. Uptake of KFE8.Tat_12.5 nanofibers by dendritic cells (DCs) was significantly increased compared with KFE8 nanofiber lacking Tat. Peritoneal and lymph node DCs of mice immunized with E.Tat_12.5 nanofibers exhibited increased presentation of the H2kb‐epitope (reminiscent for cross‐presentation) compared with DCs obtained from E. nanofiber vaccinated mice. Tetrameric and intracellular cytokine staining revealed that vaccination with E.Tat_12.5 triggered a robust and specific CD8+ T lymphocyte response, which was more pronounced than in mice vaccinated with E. nanofibers alone. Furthermore, E.Tat_12.5 nanofibers were more potent than E. nanofiber to induce antitumor immune response and tumor‐infiltrating IFN‐γ CD8 T lymphocyte. In terms of cancer vaccine development, we propose that harnessing the nanofiber‐based vaccine platform with incorporated Tat peptide could present a simple and promising strategy to induce highly effective antitumor immune response.